Neutralization of biological activity and inhibition of receptor binding by antibodies against human thrombopoietin.
Thrombopoietin (TPO) is a recently isolated cytokine that primarily regulates megakaryocytopoiesis and thrombopoiesis. We recently reported the development of a variety of antibodies (Abs) to synthetic peptides of human (h)TPO and to recombinant human TPO (rhTPO). In this study, we characterized the Abs and mapped immunologically distinct areas of the molecule. Among the five different antipeptide polyclonal Abs, only one, raised against synthetic peptide D8 to Q28, neutralized the TPO-dependent growth of FDCP-2 cells expressing human Mpl (FDCP-hMpl5 cells). One out of seven anti-rhTPO monoclonal Abs, designated as TN1, also showed neutralizing activity. TN1 was found to be specifically reactive with two proteolytic fragments, residues S1 to R117 and A60 to K122 of hTPO, indicating that the epitope(s) of TN1 is localized in residues A60 to R117 of the molecule. These two neutralizing Abs inhibited the binding of biotinylated rhTPO to FDCP-hMpl5 cells. On the other hand, the other Abs, which reacted with five polypeptides of S47 to D62, L108 to A126, N172 to A190, S262 to T284, and P306 to G332 of hTPO, did not show either the neutralizing activity or the ability to inhibit the binding of biotinylated rhTPO to the cell surface hMpl. These findings indicate that two regions, residues D8 to Q28 and A60 to R117 of hTPO, may contain the domains associated with its receptor, C-Mpl. These Abs characterized here are valuable for studying the structural analysis and the biological function of hTPO mediated by its receptor.
Tahara
T
T
Pharmaceutical Research Laboratory, Kirin Brewery Co., Ltd., Gunma, Japan.
Kuwaki
T
T
Matsumoto
A
A
Morita
H
H
Watarai
H
H
Inagaki
Y
Y
Ohashi
H
H
Ogami
K
K
Miyazaki
H
H
Kato
T
T
D000906:Antibodies; D000911:Antibodies, Monoclonal; D002455:Cell Division; D002460:Cell Line; D018604:Epitope Mapping; D006801:Humans; D009363:Neoplasm Proteins; D009500:Neutralization Tests / Q000379:methods*; D010446:Peptide Fragments; D010455:Peptides / Q000138:chemical synthesis; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D011994:Recombinant Proteins; D017384:Sequence Deletion; D013926:Thrombopoietin / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D000911:Antibodies, Monoclonal; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D010455:Peptides; D011518:Proto-Oncogene Proteins; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D011994:Recombinant Proteins; C082218:MPL protein, human; D013926:Thrombopoietin